Normal somatic cells of higher eukaryotic organisms do not proliferate indefinitely due to a process termed cellular or replicative senescence, or cellular aging. Several lines of evidence suggest that cellular senescence is a tumor suppressive mechanism that has the unselected, deleterious effect of contributing to organismic aging. Upon reaching the end of their replicative life span, cells undergo three phenotypic changes: 1) they irreversibly arrest proliferation (used here interchangeably with growth) with a G1 DNA content; 2) they become resistant to apoptotic death; 3) they show sometimes striking, cell type specific changes in differentiated functions. The growth arrest of senescent cells has been attributed to one or more critically short telomere, acquired as an inevitable consequence of multiple rounds of DNA replication by cells that lack telomerase. How a critically short telomere leads to the complex, senescent phenotype is unknown. One possibility is that a short telomere triggers a DNA damage response, in and of itself or in conjunction with intragenomic damage acquired during multiple rounds of DNA replication and cell division. The idea that DNA damage, and subsequent mutations, cause or contribute to replicative senescence and organismic aging has been long debated. This proposal aims to test this idea in ways that have not previously been possible. First, in collaboration with the Vijg Project, we will establish a new method to test mutation frequency and spectra in normal human cell cultures. We will combine viral transfer technology with plasmid-based receptor systems to deliver and evaluate mutation-reported vectors in normal and transformed human cells, cells derived from human donors with defined defects in DNA repair or age-related phenotypes, and human cells with perturbations in the establishment or maintenance of the senescent phenotype. Second, in collaboration with the Hoeijmakers Project, we will ask whether and to what extent embryo fibroblasts cultured from mice deficient or transgenic for genes known to be critical for DNA repair capacity exhibit signs of premature replicative senescence. We will establish a panel of senescent cell markers or endpoints that will enable us to compare the phenotype of mouse and human cells in culture, and relate this comparison to the phenotypes of human and mouse organisms. Third, in collaboration with both the Hoeijmakers and Vijg Projects, we will provide transgenic mice bearing a senescence-responsive element (SnRE)-reporter vector for interbreeding with DNA repair-deficient mice and mutation-reporter mice, and evaluate the senescence response of embryo fibroblasts. Together, these experiments will provide for the first time a comparison of human and mouse cells, and to a limited extent human and mouse organisms, with respect to mutation frequency and spectra, DNA repair capacity and replicative senescence. They will help critically test the relationship between DNA repair capacity, somatic mutations, replicative senescence and organismic aging.